CN105474014A - Methods for evaluating and screening S1P1 receptor agonists - Google Patents
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Abstract
It was discovered that the main effect (immunosuppressive activity) of S1P1 receptor agonists correlates with the selectivity for cells expressing a combination of S1P1 receptors and G[alpha]i2 or G[alpha]i3, and that the side effect (cardiotoxicity) of S1P1 receptor agonists correlates with the selectivity for cells expressing a combination of S1P1 receptors and G[alpha]i1.
Description
Technical field
The present invention relates to and evaluate sphingosine-1-phosphate 1 (hereinafter referred to as " the S1P1 ") immunosuppressive activity of receptor stimulating agent and the method for cardiac toxic, and screening has strong immunosuppressive activity and the method for the low S1P1 receptor stimulating agent of cardiac toxic.
Background technology
The result of numerous autoimmune diseases is, due to the immune response of exception, identify the lymphopoiesis of self mistakenly and activate, systemically attack self or attack self and specifically organize and present symptom, but its reason being varied and not clear and definite.Up to now, with suppress lymphocytic propagation, activation medicament for target, various immunodepressant is developed gradually and enters clinical practice, but the spinoff caused by nonspecific cell inhibitory effect activity, cytotoxic effect is quite a few, becomes problem.
In recent years, as the medicament for relapsing remitting multiple sclerosis disease, approved FTY720 (another name FTY-720) is not owing to being by making lymphocyte exhausted because of cell death, but carry out immunity moderation by controlling its location, therefore attracted attention as the medicament with new mechanism.But on the other hand, the serious side effects centered by cardiovascular system comprising the cardiac arrhythmia such as bradycardia, atrioventricular block (AV block) is confirmed for FTY720, thus becomes problem.(non-patent literature 1, non-patent literature 2).
The acceptor of sphingosine-1-phosphate (hereinafter referred to as " S1P ") is the G-protein conjugated receptor (GPCR) be present on cell membrane, identifiedly goes out to have 5 kinds of subtype acceptors (S1P1, S1P2, S1P3, S1P4, S1P5; Another name is endothelial differentiation gene EDG-1, EDG-5, EDG-3, EDG-6, EDG-8).FTY720 (FTY-p) and S1P1 acceptor, S1P3 acceptor, S1P4 acceptor and the S1P5 receptors bind of known phosphorylation in vivo, play function as activator.
Known lymphocyte often consumes the regular hour and cruises in blood circulation and lymphoid tissue, when moving in blood circulation from lymphoid tissue, and the extremely important effect of S1P1 receptor exerts on Lymphocyte Membrane.With FTY-p be representative S1P1 receptor stimulating agent mainly through with the S1P1 receptors bind on lymphocyte, S1P1 acceptor is taken in cell and makes the S1P1 acceptor defect on lymphocyte.Then, thus lymphocyte is isolated in secondary lymphoid tissue and circulating lymphocyte number is declined.Consequently S1P1 receptor stimulating agent plays excellent immunosuppressive activity (non-patent literature 2).On the other hand, from the research of rodent, consideration is by stimulating S1P3 acceptor to show the cardiovascular side effects such as bradycardia (non-patent literature 3,4), therefore, studying the S1P1 receptor stimulating agent of the effect reduced S1P3 acceptor.
In such situation, report as (the another name Xin Bomode of BAF312 S1P1 acceptor and S1P5 acceptor to optionally activator in recent years, Siponimod) clinical test results (non-patent literature 5), wherein as the heart rate reducing effect of spinoff with show under same amount as the circulating lymphocyte number decline effect of validity.Therefore, showing can not isolating cardiac toxicity clinically to the effect of S1P3 acceptor only by being separated, the induced cardiotoxicity showed by FTY720 be stimulate by the activator of S1P1 acceptor the toxicity caused at least partially.
S1P1 acceptor and inhibition gα protein matter (hereinafter referred to as " G α i ") coupling, in heart, by stimulating the activator of acceptor, the G α i of activation makes G-protein coupling type inward rectification type potassium channel (hereinafter referred to as " GIRK passage ") activate together with G β γ.Due in heart, the complex of GIRK1 and GIRK4 is expressed, and therefore considers to produce because this activator stimulates degradation cardiac toxic (non-patent literature 5,6) under AV block, heart rate.
About the agonist activity of the S1P as the endogenous agonist to S1P acceptor, although report different according to evaluation assessment and have some differences, usually show 50% activation concentration (EC50 value) of about about 10nM.On the other hand, although the blood level of the general strong ordinary person of S1P exists with the high concentration of the decades of times of the such EC50 value of hundreds of nM, in general strong ordinary person, cardiac toxic is not brought out.And then, there is following contradiction: although in the same manner as BAF312 with FTY-p showing the EC50 value of same degree effectively blood level be the concentration that tens of nM is low like this, performance cardiac toxic.
Like this, the mechanism of the immunosuppressive activity of S1P receptor stimulating agent performance as its main effect, the cardiac toxic as its spinoff is illustrated at present not yet fully.
Prior art document
Patent documentation
Patent documentation 1:WO2013/094761
Non-patent literature
Non-patent literature 1:Science, 296,346-349 (2002)
Non-patent literature 2:JournaloftheAmericanSocietyofNephrology, 13 (4), 1073-1083 (2002)
Non-patent literature 3:JournalofBiologicalChemistry, 279 (14), 13839-13848 (2004)
Non-patent literature 4:MolecularPharmacology, 58,449-454 (2000)
Non-patent literature 5:BritishJournalofPharmacology, 167,1035-1047 (2012)
Non-patent literature 6:JournalofBiologicalChemistry, 276 (8), 5505-5510 (2001)
Non-patent literature 7:NatureChemicalBiology, 8,622-630 (2012)
Summary of the invention
Invent problem to be solved
The present invention makes in view of such situation, its object is to, and illustrates the mechanism that S1P receptor stimulating agent plays main effect and spinoff, based on the information of this mechanism, provides and evaluates the main effect of S1P receptor stimulating agent and the method for spinoff.And then, the object of the invention is to, based on the information of this mechanism, provide screening to have strong main effect and the method for the S1P receptor stimulating agent of few side effects.
For solving the method for problem
There is numerous molecule relevant to the signal transduction of S1P1 acceptor, in these molecules, even if be conceived to the gα protein with S1P1 coupled receptors, also there is numerous hypotypes.In such a case, present inventor conducts in-depth research to solve above-mentioned problem, found that, the immunosuppressive activity of S1P1 receptor stimulating agent is relevant to the selectivity of cell of the combination expressing S1P1 acceptor and G α i2 or G α i3, and the cardiac toxic of S1P1 receptor stimulating agent is relevant to the selectivity of cell of the combination expressing S1P1 acceptor and G α i1.That is, present inventor's final certification is with G α i1, G α i2 and the G α i3 of S1P1 coupled receptors, relevant to the immunosuppressive activity of S1P receptor stimulating agent, cardiac toxic.
And, based on this opinion, present inventor finds, by detecting G α subunit (G α i1, G α i2, G α i3) the specific agonist activity of coupling, the intensity of the immunosuppressive activity of S1P1 receptor stimulating agent, the height of cardiac toxic can be evaluated, and then the strong and S1P1 receptor stimulating agent that cardiac toxic is low of immunosuppressive activity can be screened, thus complete the present invention.
Therefore, the present invention relates to evaluation method and the screening technique of S1P1 receptor stimulating agent, it is characterized in that, detect the agonist activity to S1P1 acceptor of the G α ac subunit specificity of coupling, in more detail, provide following (1) ~ (9).
(1) method of the intensity of the immunosuppressive activity of S1P1 receptor stimulating agent is evaluated,
The method using the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i2 coupling and and G α i3 coupling S1P1 acceptor in the agonist activity of at least one as index.
(2) method of the intensity of the immunosuppressive activity of S1P1 receptor stimulating agent is evaluated,
The method comprise the S1P1 acceptor that measures S1P1 receptor stimulating agent pair and G α i2 coupling and and the operation of agonist activity of S1P1 acceptor of G α i3 coupling,
When S1P1 receptor stimulating agent pair and G α i2 coupling S1P1 acceptor and and the agonist activity of S1P1 acceptor of G α i3 coupling higher than agonist activity to the S1P1 acceptor with G α i1 coupling, be evaluated as S1P1 receptor stimulating agent and can play strong immunosuppressive activity.
(3) method of the intensity of the immunosuppressive activity of S1P1 receptor stimulating agent is evaluated,
The method comprise the S1P1 acceptor that measures S1P1 receptor stimulating agent pair and G α i2 coupling and and the operation of agonist activity of S1P1 acceptor of G α i3 coupling,
When S1P1 receptor stimulating agent pair and G α i2 coupling S1P1 acceptor and and G α i3 coupling S1P1 acceptor in the agonist activity of at least one higher than endogenous agonist S1P, be evaluated as S1P1 receptor stimulating agent and can play strong immunosuppressive activity.
(4) method that S1P1 receptor stimulating agent produces the easy degree of cardiac toxic is evaluated,
The method is using the agonist activity of the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling as index.
(5) method that S1P1 receptor stimulating agent produces the easy degree of cardiac toxic is evaluated,
The method comprises the operation of the agonist activity of the S1P1 acceptor measuring S1P1 receptor stimulating agent pair and G α i1 coupling,
When the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling agonist activity lower than to the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling, be evaluated as S1P1 receptor stimulating agent and not easily produce cardiac toxic.
(6) method that S1P1 receptor stimulating agent produces the easy degree of cardiac toxic is evaluated,
The method comprises the operation of the agonist activity of the S1P1 acceptor measuring S1P1 receptor stimulating agent pair and G α i1 coupling,
When the agonist activity of the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling is lower than endogenous agonist S1P, is evaluated as S1P1 receptor stimulating agent and not easily produces cardiac toxic.
(7) screening technique of S1P1 receptor stimulating agent,
The method comprises:
Operation (a), measure test compound pair and the S1P1 acceptor of G α i1 coupling and the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling, and
Operation (b), select to the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling higher than the compound of the agonist activity to the S1P1 acceptor with G α i1 coupling.
(8) method Gen Ju (7), in operation (b), select to the S1P1 acceptor of G α i2 coupling and and the compound of the agonist activity comparison of S1P1 acceptor of G α i3 coupling and high more than 30 times of the agonist activity of the S1P1 acceptor of G α i1 coupling.
(9) screening technique of S1P1 receptor stimulating agent,
The method comprises:
Operation (a), measure test compound pair and the S1P1 acceptor of G α i1 coupling and the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling, and
Operation (b), select to the agonist activity of the S1P1 acceptor with G α i1 coupling lower than endogenous agonist S1P and to the S1P1 acceptor of G α i2 coupling and and G α i3 coupling S1P1 acceptor in the agonist activity of at least one higher than the compound of endogenous agonist S1P.
In addition, about " S1P1 acceptor " in the present invention, on ncbi database, its representative amino acid sequence is registered as NP001391.2, and the base sequence of corresponding mRNA is registered as NM001400.4.
In addition, about " G α i1 (gene name is GNAI1) ", on ncbi database, the amino acid sequence of its representative is registered as NP_002060.4, and the base sequence of corresponding mRNA is registered as NM002069.5.
In addition, about " G α i2 (gene name is GNAI2) ", on ncbi database, the amino acid sequence of its representative is registered as NP_002061.1, and the base sequence of corresponding mRNA is registered as NM002070.2.
In addition, about " G α i3 (gene name is GNAI3) ", on ncbi database, the amino acid sequence of its representative is registered as NP_006487.1, and the base sequence of corresponding mRNA is registered as NM006496.3.
The effect of invention
In the evaluation method of agonist activity in the past, the main effect of S1P1 receptor stimulating agent (validity) and spinoff difference can not be come and evaluate.In the present invention, by being divided into the agonist activity detected with the hypotype of the gα protein of S1P1 coupled receptors (G α i1, G α i2, G α i3) S1P1 acceptor, thus the main effect of S1P1 receptor stimulating agent and spinoff difference can being come and evaluate.In addition, excellent S1P1 receptor stimulating agent can effectively be screened thus.
Embodiment
< evaluates the method > of the intensity of the immunosuppressive activity of S1P1 receptor stimulating agent
The invention provides the method for the intensity of the immunosuppressive activity evaluating S1P1 receptor stimulating agent.Find in the present embodiment, the intensity of the immunosuppressive activity of S1P1 receptor stimulating agent to the S1P1 acceptor of G α i2 coupling or relevant with the agonist activity of S1P1 acceptor of G α i3 coupling.Therefore, evaluation method of the present invention be using the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i2 coupling and and G α i3 coupling S1P1 acceptor in the agonist activity of at least one as referring to calibration method.
For " immunosuppressive activity " in the present invention, such as, can control using the decline of circulating lymphocyte number as index.For circulating lymphocyte number, such as, can be measured by the method recorded in the present embodiment 2.
The mensuration of the agonist activity of the hypospecificity of the G α i in the present invention can be passed through such as, and the method (method recorded in non-patent literature 7) recorded in the present embodiment 1 (2) is carried out.That is, make S1P1 acceptor and specific G alpha hypotype and G γ 2 at cells together with G β 1, using the separation rate of the gα protein caused by S1P1 receptor stimulating agent and G β γ albumen as index, can agonist activity be measured.
In addition, if known activator usually stimulates S1P1 acceptor, then in cell, cAMP amount is regulated by the direction of reducing.Therefore, such as, use the cell of coexpression S1P1 acceptor and specific G α i hypotype, using the minimizing of measuring at the cAMP being made by forskolin etc. under the condition that in cell, cAMP amount rises, caused by S1P1 receptor stimulating agent as index, also can measure the agonist activity of G α i hypospecificity.
In addition, if cytomorphosis, then the resistance value formed on monofilm changes.Knownly utilize this phenomenon, film is expressed with monolayer cultivation the cell of S1P acceptor, measure the method (InvestOphthalmolVisSci.2005Jun of the change of the resistance value produced owing to adding S1P1 receptor stimulating agent; 46 (6): 1927-33.).Based on the method, such as, film is cultivated the cell of coexpression S1P1 acceptor and specific G alpha hypotype, using the change of the resistance value produced owing to adding S1P1 receptor stimulating agent as index, also can measure the agonist activity of G α i hypospecificity.
In addition, usually the known cell membrane of expression GPCR, compound and γ-[35S] GTP that makes reacts in damping fluid, quantitative method (LifeScienceVolume74 is carried out using the amount being combined in the marked body on film as the activity value of activator, Issue4,12December2003, Pages489-508).Therefore, such as, use the cell membrane of coexpression S1P1 acceptor and specific G α i hypotype, using because GTP exchange reaction γ-[35S] GTP is to the accumulation of membrane portions as index, also can measure the agonist activity of G α i hypospecificity.
In a kind of mode of the present invention, measure S1P1 receptor stimulating agent pair and G α i2 coupling S1P1 acceptor and and the agonist activity of S1P1 acceptor of G α i3 coupling, when S1P1 receptor stimulating agent pair and G α i2 coupling S1P1 acceptor and and the agonist activity of S1P1 acceptor of G α i3 coupling higher than agonist activity to the S1P1 acceptor with G α i1 coupling, be evaluated as S1P1 receptor stimulating agent and can play strong immunosuppressive activity.
Preferably to the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling, be more than 10 times of the agonist activity to the S1P1 acceptor with G α i1 coupling, be more preferably more than 20 times, preferably more than 30 times further.
In addition, in another way of the present invention, measure S1P1 receptor stimulating agent pair and G α i2 coupling S1P1 acceptor and and the agonist activity of S1P1 acceptor of G α i3 coupling, when S1P1 receptor stimulating agent pair and G α i2 coupling S1P1 acceptor and and G α i3 coupling S1P1 acceptor in the agonist activity of at least one higher than endogenous agonist S1P, be evaluated as S1P1 receptor stimulating agent and can play strong immunosuppressive activity.
The agonist activity becoming the endogenous agonist S1P of contrast can measure when measuring the agonist activity of S1P1 receptor stimulating agent, also can use the value measured in advance.Such as, when utilizing the method (method recorded in non-patent literature 7) recorded in the present embodiment 1 (2) to measure, when the EC50 value (nM) of the agonist activity to the S1P1 acceptor with G α i2 coupling be the situation of less than 14 or be less than 8 to the EC50 value (nM) of the agonist activity of the S1P1 acceptor with G α i3 coupling, be evaluated as S1P1 receptor stimulating agent and can play strong immunosuppressive activity.
< evaluates the method > that S1P1 receptor stimulating agent produces the easy degree of cardiac toxic
The invention provides and evaluate the method that S1P1 receptor stimulating agent produces the easy degree of cardiac toxic.Find in the present embodiment, the easy degree that the cardiac toxic of S1P1 receptor stimulating agent produces is relevant to the agonist activity of the S1P1 acceptor with G α i1 coupling.Therefore, evaluation method of the present invention is using the agonist activity of the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling as index.
" cardiac toxic " in the present invention can by such as, and AV block, heart rate decline and control as index.AV block can pass through such as, and the method recorded in the present embodiment 3 measures.In addition, the method for measuring of the agonist activity of the hypospecificity of the G α i in the present invention is described above.
In a kind of mode of the present invention, measure the agonist activity of the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling, when the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling agonist activity lower than to the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling, be evaluated as S1P1 receptor stimulating agent and not easily produce cardiac toxic.
Preferably to the agonist activity of the S1P1 acceptor with G α i1 coupling be to the S1P1 acceptor of G α i2 coupling and and G α i3 coupling S1P1 acceptor agonist activity less than 1/10th, be more preferably less than 1/20th, more preferably less than 1/30th.
In addition, in another way of the present invention, measure the agonist activity of the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling, when the agonist activity of the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling is lower than endogenous agonist S1P, is evaluated as S1P1 receptor stimulating agent and not easily produces cardiac toxic.
The agonist activity of endogenous agonist S1P in contrast can measure when measuring the agonist activity of S1P1 receptor stimulating agent, also can use the value measured in advance.Such as, when utilizing the method (method recorded in non-patent literature 7) recorded in the present embodiment 1 (2) to measure, when the agonist activity to the S1P1 acceptor with G α i1 coupling EC50 value (nM) higher than 261, be evaluated as S1P1 receptor stimulating agent and not easily produce cardiac toxic.
The screening technique > of <S1P1 receptor stimulating agent
The invention provides the screening technique of S1P1 receptor stimulating agent.In the present embodiment, specify that the immunosuppressive activity of S1P1 receptor stimulating agent is relevant to the selectivity of cell of the combination expressing S1P1 acceptor and G α i2 or G α i3, the cardiac toxic of S1P1 receptor stimulating agent is relevant to the selectivity of cell of the combination expressing S1P1 acceptor and G α i1.Screening technique of the present invention utilizes so relevant method.
Test compound for screening is not particularly limited, such as, synthesis low molecular compound library, the expression product of gene library, peptide library, antibody, bacterium h substance, the extract of cell (microorganism, vegetable cell, zooblast) and culture supernatant, purifying or partial purification polypeptide, sea life, the extract being derived from plant or animal, phage display random peptide library can be used.In addition, the method detecting the agonist activity of the hypospecificity of G α i is described above.
In a kind of mode of the present invention, measure test compound pair and the S1P1 acceptor of G α i1 coupling and the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling, select to the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling higher than the compound of the agonist activity to the S1P1 acceptor with G α i1 coupling.
Preferred selection to the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling higher than the compound of the agonist activity more than 10 times to the S1P1 acceptor with G α i1 coupling, more preferably select the compound of high more than 20 times, preferably select the compound of high more than 30 times further.
In addition, in another way of the present invention, measure test compound pair and the S1P1 acceptor of G α i1 coupling and the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling, select to the agonist activity of the S1P1 acceptor with G α i1 coupling lower than endogenous agonist S1P and to the S1P1 acceptor of G α i2 coupling and and G α i3 coupling S1P1 acceptor in the agonist activity of at least one higher than the compound of endogenous agonist S1P.
The agonist activity of endogenous agonist S1P in contrast can measure when measuring the agonist activity of S1P1 receptor stimulating agent, also can use the value measured in advance.Such as, when utilizing the method (method recorded in non-patent literature 7) recorded in the present embodiment 1 (2) to measure, can to select the EC50 value (nM) of the agonist activity of the S1P1 acceptor with G α i1 coupling be less than 14 higher than 261 and to the EC50 value (nM) of the agonist activity of the S1P1 acceptor with G α i2 coupling or be the compound of less than 8 to the EC50 value (nM) of the agonist activity of the S1P1 acceptor with G α i3 coupling.Consider that the compound selected like this shows strong immunosuppressive activity, and cardiac toxic is low.
Embodiment
Based on embodiment, more specific description is carried out to the present invention below, but the present invention does not limit by following embodiment.
[embodiment 1] S1P1 receptor stimulating agent immunosuppressive activity is evaluated
(1) evaluation of common S1P1 receptor agonist activity
As the method for common evaluation S1P1 receptor agonist activity, the known Chinese hamster ovary celI etc. using expression S1P1 acceptor, using the combination to acceptor, by combining the active method (patent documentation 1) measuring agonist activity as index of the signal transduction produced.According to the method recorded in the test example 1 of patent documentation 1, determine the S1P1 receptor agonist activity of the compound (hereinafter referred to as " compoundB (compd B) ") recorded in the compound (hereinafter referred to as " compoundA (compd A) ") and patent documentation 1-embodiment 2 recorded in the example48 (embodiment 48) of S1P, FTY-p, BAF312, No. 1826197th, European patent.Result S1P1 receptor agonist activity is 6.4nM, 0.08nM, 3.0nM, 0.3nM, 9.2nM respectively.
(2) evaluation of the S1P1 receptor agonist activity of the hypospecificity of gα protein
Then, according to the method recorded in non-patent literature 7, the evaluation of the S1P1 receptor agonist activity of the hypospecificity of gα protein has been carried out.In addition, expression condition is according to " SupplementaryMethods (compensation process) " item of non-patent literature 7, and condition determination is according to " Bioluminescenceresonanceenergytransfermeasurement (Bioluminescence Resonance Energy transfer assay) " item.
First, make HEK293T cell coexpression S1P1 acceptor, specific G alpha hypotype (G α i1, G α i2 or G α i3), G γ 2 and G β 1, using the separation rate of the gα protein caused by endogenous agonist S1P and G β γ albumen as index, determine agonist activity.Consequently, 50% activation concentration (EC50 value) of S1P is 261nM under G α i1 expression condition, and be 14.6nM under G α i2 expression condition, be 8.4nM under G α i3 expression condition.
Then, under same test condition, the EC50 value of FTY-p, BAF312, compoundA and compoundB is 17.6nM, 122nM, 98nM, 640nM respectively under G α i1 expression condition; About 100nM, 81nM, 11nM, 6nM respectively under G α i2 expression condition; 0.94nM, 27nM, 85nM, 19nM (table 1) respectively under G α i3 expression condition.
Table 1
| S1P1-Gαi1 | S1P1-Gαi2 | S1P1-Gαi3 | |
| S1P | 261 | 14.6 | 8.4 |
| FTY-p | 17.6 | 100 | 0.94 |
| BAF312 | 122 | 81 | 27 |
| Compound A | 98 | 11 | 85 |
| Compound B | 640 | 6 | 19 |
Distinguish from this result, FTY-p, BAF312 and compoundA are strong to the activity of G α i1, and low to the selectivity of G α i2, G α i3.Distinguish on the other hand, the activity of compoundB to G α i1 is weak, high to the selectivity of G α i2 and G α i3.Can say that compoundB is the strong deflection activator (biasedagonist) making more to offset in the deflection of the activity of S1P display.
[embodiment 2] S1P1 receptor stimulating agent efficiency evaluation
Give BAF312 or compoundB with different consumptions to male SD rat per os, utilize the blood lymphocyte number of determination of blood cell device mensuration after 3,6 hours and after 24 hours.Result is all suppress lymphocyte number after giving 6 hours, until 24 hours still observe its effect the most by force.
Suppress consumption (ED50) about 50% lymphocyte number after 6 hours and after 24 hours, BAF312 is 0.1mg/kg, >1mg/kg respectively, and compoundB is 0.17mg/kg, 0.65mg/kg respectively.Calculate the effective blood level after 24 hours, result BAF312 is >208nM, compoundB is 176nM.
[embodiment 3] S1P1 receptor stimulating agent cardiac toxic is evaluated
The compoundB of the effective blood level using display and BAF312 almost equal, the cardiac toxic expression intensity in the lower guinea pig heart toxicity assessment model of research anesthesia.This model uses the cavy high to the responsiveness of S1P1 acceptor, and is the model that strongly can detect the cardiac toxic of S1P1 receptor-independent by carrying out under anaesthesia.Detecting ECG instrument and pace-making instrument are installed to the male guinea pig having carried out artificial respiration management under anaesthesia, carries out medicament and give.
Consequently, when within 10 minutes, infusion gives the BAF312 of 0.01mg/kg, in 4 examples, 4 examples express completeness AV block, until all cases all continue completeness AV block after 1 hour.
On the other hand, when giving compoundB under identical condition, in 4 examples, 1 example does not all express completeness AV block.
In addition, the blood level at the end of BAF312 and compoundB administration is 17nM, 91nM respectively, and the blood level after 1 hour is <4nM, 27nM respectively.This fact shows, although the active stronger compoundB blood level of deflection is higher, is more weak cardiac toxic.
Utilizability in industry
According to the present invention, the main effect of S1P1 receptor stimulating agent (validity) and spinoff difference can be come and evaluate, effectively can screen excellent S1P1 receptor stimulating agent.Therefore, the present invention can make large contribution in the exploitation of medicine and evaluation field.
Claims (9)
1. evaluate the method for the intensity of the immunosuppressive activity of S1P1 receptor stimulating agent,
The method using the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i2 coupling and and G α i3 coupling S1P1 acceptor in the agonist activity of at least one as index.
2. evaluate the method for the intensity of the immunosuppressive activity of S1P1 receptor stimulating agent,
The method comprise the S1P1 acceptor that measures S1P1 receptor stimulating agent pair and G α i2 coupling and and the operation of agonist activity of S1P1 acceptor of G α i3 coupling,
When S1P1 receptor stimulating agent pair and G α i2 coupling S1P1 acceptor and and the agonist activity of S1P1 acceptor of G α i3 coupling higher than agonist activity to the S1P1 acceptor with G α i1 coupling, be evaluated as S1P1 receptor stimulating agent and can play strong immunosuppressive activity.
3. evaluate the method for the intensity of the immunosuppressive activity of S1P1 receptor stimulating agent,
The method comprise the S1P1 acceptor that measures S1P1 receptor stimulating agent pair and G α i2 coupling and and the operation of agonist activity of S1P1 acceptor of G α i3 coupling,
When S1P1 receptor stimulating agent pair and G α i2 coupling S1P1 acceptor and and G α i3 coupling S1P1 acceptor in the agonist activity of at least one higher than endogenous agonist S1P, be evaluated as S1P1 receptor stimulating agent and can play strong immunosuppressive activity.
4. evaluate the method that S1P1 receptor stimulating agent produces the easy degree of cardiac toxic,
The method is using the agonist activity of the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling as index.
5. evaluate the method that S1P1 receptor stimulating agent produces the easy degree of cardiac toxic,
The method comprises the operation of the agonist activity of the S1P1 acceptor measuring S1P1 receptor stimulating agent pair and G α i1 coupling,
When the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling agonist activity lower than to the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling, be evaluated as S1P1 receptor stimulating agent and not easily produce cardiac toxic.
6. evaluate the method that S1P1 receptor stimulating agent produces the easy degree of cardiac toxic,
The method comprises the operation of the agonist activity of the S1P1 acceptor measuring S1P1 receptor stimulating agent pair and G α i1 coupling,
When the agonist activity of the S1P1 acceptor of S1P1 receptor stimulating agent pair and G α i1 coupling is lower than endogenous agonist S1P, is evaluated as S1P1 receptor stimulating agent and not easily produces cardiac toxic.
7.S1P1 the screening technique of receptor stimulating agent,
The method comprises:
Operation (a), measure test compound pair and the S1P1 acceptor of G α i1 coupling and the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling, and
Operation (b), select to the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling higher than the compound of the agonist activity to the S1P1 acceptor with G α i1 coupling.
8. method according to claim 7, in operation (b), select to the S1P1 acceptor of G α i2 coupling and and the compound of the agonist activity comparison of S1P1 acceptor of G α i3 coupling and high more than 30 times of the agonist activity of the S1P1 acceptor of G α i1 coupling.
9.S1P1 the screening technique of receptor stimulating agent,
The method comprises:
Operation (a), measure test compound pair and the S1P1 acceptor of G α i1 coupling and the S1P1 acceptor of G α i2 coupling and and the agonist activity of S1P1 acceptor of G α i3 coupling, and
Operation (b), select to the agonist activity of the S1P1 acceptor with G α i1 coupling lower than endogenous agonist S1P and to the S1P1 acceptor of G α i2 coupling and and G α i3 coupling S1P1 acceptor in the agonist activity of at least one higher than the compound of endogenous agonist S1P.
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| CN101663297A (en) * | 2007-04-19 | 2010-03-03 | 葛兰素集团有限公司 | Oxadiazole substituted indazole derivatives for use as sphingosine 1-phosphate (s1p) agonists |
| WO2013094761A1 (en) * | 2011-12-23 | 2013-06-27 | Ｍｅｉｊｉ Ｓｅｉｋａファルマ株式会社 | Novel s1p receptor modulator |
| WO2013109543A1 (en) * | 2012-01-20 | 2013-07-25 | The Board Of Trustees Of The Leland Stanford Junior University | Small molecule cmklr1 antagonists in demyelinating disease |
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| EP2592066B1 (en) | 2004-12-13 | 2014-12-03 | Ono Pharmaceutical Co., Ltd. | Aminocarboxylic acid derivative and medical use thereof |
| WO2008128951A1 (en) | 2007-04-19 | 2008-10-30 | Glaxo Group Limited | Oxadiazole substituted indazole derivatives for use as sphingosine 1-phosphate (s1p) agonists |
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2014
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- 2014-08-20 EP EP14838211.2A patent/EP3037818B1/en not_active Expired - Fee Related
- 2014-08-20 ES ES14838211T patent/ES2727516T3/en active Active
- 2014-08-20 WO PCT/JP2014/071737 patent/WO2015025871A1/en active Application Filing
- 2014-08-20 KR KR1020167000333A patent/KR20160043948A/en not_active Application Discontinuation
- 2014-08-20 JP JP2015532873A patent/JP6450315B2/en not_active Expired - Fee Related
- 2014-08-20 AU AU2014309817A patent/AU2014309817A1/en not_active Abandoned
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| WO1997048820A1 (en) * | 1996-06-21 | 1997-12-24 | Aurora Biosciences Corporation | Promiscuous g-protein compositions and their use |
| CN1592625A (en) * | 2001-08-10 | 2005-03-09 | 惠氏公司 | G protein-coupled receptor assay |
| JP2003210192A (en) * | 2002-01-18 | 2003-07-29 | Sumitomo Pharmaceut Co Ltd | Method for screening ligand for orphan gpcr |
| CN1838961A (en) * | 2003-05-30 | 2006-09-27 | 塞诺菲-安万特德国有限公司 | Use of s1p |
| CN101663297A (en) * | 2007-04-19 | 2010-03-03 | 葛兰素集团有限公司 | Oxadiazole substituted indazole derivatives for use as sphingosine 1-phosphate (s1p) agonists |
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| WO2013109543A1 (en) * | 2012-01-20 | 2013-07-25 | The Board Of Trustees Of The Leland Stanford Junior University | Small molecule cmklr1 antagonists in demyelinating disease |
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| EP3037818B1 (en) | 2019-03-27 |
| JPWO2015025871A1 (en) | 2017-03-02 |
| EP3037818A1 (en) | 2016-06-29 |
| CA2919980A1 (en) | 2015-02-26 |
| US20160195513A1 (en) | 2016-07-07 |
| US9885703B2 (en) | 2018-02-06 |
| CN105474014B (en) | 2017-04-05 |
| EP3037818A4 (en) | 2017-01-11 |
| JP6450315B2 (en) | 2019-01-09 |
| ES2727516T3 (en) | 2019-10-16 |
| WO2015025871A1 (en) | 2015-02-26 |
| AU2014309817A1 (en) | 2016-02-18 |
| KR20160043948A (en) | 2016-04-22 |
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